Celiac disease (CD) is an autoimmune disease leading to gastrointestinal symptoms and mineral deficiencies. The pathogenetic mechanisms, besides the clear HLA association, are elusive. Among environmental factors infections have been proposed. Covid-19 infection results in a systemic inflammatory response that often also involves the gastrointestinal tract. The aim of the present study was to investigate whether Covid-19 infection could increase the risk for CD.All patients, both children and adults, in the county Skåne (1.4 million citizens) in southern Sweden with newly diagnosed biopsy- or serology-verified CD or a positive tissue transglutaminase antibody test (tTG-ab) during 2016–2021 were identified from registries at the Departments of Pathology and Immunology, respectively. Patients with a positive Covid-19 PCR or antigen test in 2020 and 2021 were identified from the Public Health Agency of Sweden.During the Covid-19 pandemic (March 2020 – December 2021), there were 201 050 cases of Covid-19 and 568 patients with biopsy- or serology-verified CD or a first-time positive tTG-ab tests, of which 35 patients had been infected with Covid-19 before CD. The incidence of verified CD and tTG-ab positivity was lower in comparison to before the pandemic  -3.0, 95% CI -5.7 – -0.3, p = 0.028). The incidence of verified CD and tTG-ab positivity in patients with and without prior Covid-19 infection was 21.1 and 22.4 cases per 100 000 person-years, respectively .Our results indicate that Covid-19 is not a risk factor for CD development. While gastrointestinal infections seem to be an important part of the CD pathogenesis, respiratory infections probably are of less relevance. Celiac disease (CD) is a chronic autoimmune disorder triggered by the consumption of gluten in genetically predisposed individuals . The incDietary gluten consumption and genetical predisposition in the form of the HLA-DQ2 or -DQ8 alleles are seen in almost all CD cases. It is estimated that around 50% of the Swedish population carry the HLA-DQ2 or -DQ8 alleles , but onlThere is growing evidence that infections early in life increase the risk of CD, as shown in both retrospective and prospective observational studies –12. The Coronavirus disease-19 (Covid-19) was declared a pandemic by WHO in March 2020. As of 31 Jan 2023, there has been over 753 million cases of Covid-19 worldwide . The virDuring a pandemic many individuals are infected almost simultaneously and with varying disease courses. The Covid-19 pandemic gives a unique opportunity to study whether a new virus that has never infected humanity before might have an impact on the onset of an immune driven disease. Consequently, we aimed to investigate whether Covid-19 infection could increase the risk of CD.A retrospective observational cohort study comprising both children and adults was designed to investigate if Covid-19 infection affects the risk of subsequent CD. All cases of CD between 2016 and 2021 and Covid-19 between 2020 and 2021 in Region Skåne, a county in southern Sweden, were identified. There were 1.32 million residents in the region in 2016 and 1.40 million in 2021 . All resBiopsy-verified CD cases were retrieved from the registries at the pathology departments in Region Skåne. To identify CD cases, Systematized Nomenclature of Medicine (SNOMED) clinical terms D6218 (celiac disease), M58005  and M58006  were used. M58007  is not used in Region Skåne. The SNOMED topography codes in the database search were T64 (duodenum) and T65 (jejunum/ileum). Due to variations in time interval between the arrival and examination of the specimen, the biopsy’s arrival date to the pathology department  was used as the date of CD diagnosis. To exclude control biopsies during follow-up all biopsies fulfilling the above criteria between 2010 and 2021 were identified and the date of the first biopsy indicating CD was used as the date of diagnosis. The use of villous atrophy in biopsy reports from Swedish pathology departments has been validated previously, with a specificity of 95% for a clinical CD diagnosis .Furthermore, patients with a positive IgA or IgG tissue transglutaminase antibody (tTG-ab) serology were identified. tTG-ab titers were analyzed at the Department of Clinical immunology at Skåne University Hospital according to established procedures. Levels of tTG-ab > 10 kilo-arbitrary units per liter (kU/L) were considered positive. Patients with a positive tTG-ab serology before the start of the study were excluded.A verified CD diagnosis was based on either a biopsy with villous atrophy or a serology-based diagnosis, defined as two separate tTG-ab tests more than 10 times above the upper limit of normal. This is in accordance with the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) criteria from 2012 for serology-based diagnosis in children  and fromSeparately, an additional analysis during the period 2016-21 was performed in which patients with a first-time positive tTG-ab test was included regardless of biopsy status. To be able to identify both onset of manifest CD or any increase in tTG-ab-status a combined outcome was used, i.e., presence of either a biopsy indicating CD or a first positive tTG-ab (above the reference cut-off 10 kU/L).It was mandatory for all healthcare providers, both public and private, to report all patients that tested positive for Covid-19 during the pandemic to the Public Health Agency of Sweden. The Covid-19 cases in Region Skåne from 2020 to 2021 were retrieved from this registry. If a patient had several positive tests, the first positive test was used as the date of Covid-19 diagnosis for our analyses. SARS-CoV-2 RNA was detected using polymerase chain reaction (PCR) or antigen tests. The PCR was based on the method developed by Corman et al. .ir command and tested using Chi2. Groupwise comparisons for baseline characteristics were done with Chi2 for binary data and Student’s T-test for continuous outcomes that were normally distributed. Normal distribution was checked graphically and with calculation of skewness. Statistical analyses were performed with Stata Statistical Software 17.0 for Windows . A p-value < 0.05 was considered significant in all analyses.Incidence calculations were carried out based on monthly population data from Statistics Sweden . CompariThe study was approved by the Swedish Ethical Review Authority (protocol number 2021–04648). Since the present study was strictly register-based informed consent was not required. Waiver of informed consent was approved by the Swedish Ethical Review Authority. All methods used in this study were carried out in accordance with relevant guidelines and regulations.To get an overview of the CD epidemiology in the region, the yearly incidence of the disease from 2016 to 2021 was determined. There were 222 cases of biopsy- or serology-verified CD in 2016 and 193 cases in 2021. The incidence declined during the period, from 16.8 in 2016 to 13.8 cases per 100 000 person-years in 2021 (incidence rate difference (IRD) -3.0, 95% confidence interval (CI) -5.9 – -0.5, p = 0.046; Fig. The monthly incidence of the combined outcome (verified CD or tTG-ab positivity) during the Covid-19 pandemic was determined, from March 2020 to December 2021, and compared with the same time before the pandemic (May 2018 to February 2020). In total, there were 639 cases before and 573 cases during the pandemic. Of the patients, 62% and 58% were women before and during the pandemic, respectively (p = 0.085). They had a mean age of 25.7 years (range 0.9–93.7 years) and 24.6 years (range 0.8–87.8 years), respectively . The incidence decreased from 25.5 cases per 100 000 person-years before the pandemic to 22.5 during the pandemic  and 51% were women. There were 568 cases of verified CD or tTG-ab positivity during this time. Of these, 35 patients were infected with Covid-19 before CD or tTG-ab positivity. The incidence in patients with previous Covid-19 infection was 21.1 cases per 100 000 person-years. Among patients who got CD or tTG-ab positivity without previously being diagnosed with Covid-19, the incidence was 22.4 cases per 100 000 person-years. There was no difference between the two groups (IRD − 1.3 cases per 100 000 person-years, 95% CI -8.5–5.9, p = 0.75; Table The group who developed verified CD or tTG-ab positivity after Covid-19 infection did not differ in gender compared to those without a previous Covid-19 infection. There were 57% females in both groups Table . Those wThere is growing evidence that infections may constitute an important part of CD pathogenesis –21. LessThe decreased incidence of CD could be due to reduced accessibility to healthcare during the pandemic, but the fact that the decline started several years before the pandemic makes this less likely. A reduced access to invasive procedures such as gastroscopy could be expected, but we also found a decrease in positive tTG-ab cases. Since the proportion of tests with a positive outcome remained unchanged there is no indication that any increase in tTG-ab positivity did occur. The incidence of CD in Sweden started to decline already before the start of our study period . ConsequA German study by Lingel et al.  in adultWhile several studies report that gastroenteritis in children seems to trigger CD , 13–17, We did not have any information about Covid-19 vaccination status in the study. The majority of Swedish citizens received the first vaccine dose during early summer 2021 , a minorOur results are strengthened by the rather large cohort of 1.4 million citizens. Sweden’s strategy to mitigate the Covid-19 spread and protect the elderly, instead of large-scale lockdowns, may have resulted in a rather large proportion of Covid-19 infections which theoretically could have increased the chance of finding an effect in our study . By incla priori power analysis it was estimated that, given a baseline incidence around 55 CD cases per 100 000 person-years [Remarkably, in this cohort of 1.4 million citizens, of whom more than 200 000 had been diagnosed with Covid-19, only 35 patients got verified CD or tTG-ab positivity after the Covid-19 infection. In an on-years  and an oon-years , a cohorMost previous investigations on the association between infections and CD have studied young children and rarely adults –20. SincAnother potential limitation is the fact that the follow-up time could be too short. The exact timing of CD onset following an infection is unknown. Whereas Kårhus et al.  reportedIt is not known to which extent patients might have had Covid-19 without being tested. During the first half of 2020 testing was focused on people at increased risk of severe Covid-19 such as elderly and patients requiring hospital care, while testing was less available for the general population. Testing was also restricted due to shortage of testing equipment. During the latter half of 2020 and onwards, testing was recommended for the general population . AlthougOur results indicate that Covid-19 is not a risk factor for CD development. While gastrointestinal infections seem to be an important part of the CD pathogenesis, respiratory infections probably are of less relevance. Although there are some limitations, this study sheds some important light on the issue. There is a need for further research to confirm our results. Such studies should be case-controlled to account for differences in age among patients with CD and Covid-19. Still, there will be an issue of Covid-19 underdiagnosis due to varying accessibility to testing during the pandemic. Ideally, further studies would be prospective to identify more Covid-19 cases and to ensure that Covid-19 preceded CD development. Possible differences in children and adults should also be taken into consideration.


In a retrospective study, researchers analyzed data from 909 patients with papillary thyroid carcinoma (PTC) who underwent thyroidectomy and neck dissection. The aim was to validate the relationship between lymph node ratio (LNR) and recurrence in patients with fewer than six lymph nodes examined after surgery. Over an average follow-up of 127.24 ± 33.6 months, recurrences occurred in 5.1% of the patients. The analysis identified an LNR ≥ 0.29 as an independent prognostic factor for recurrence. This means that patients with an LNR equal to or higher than 0.29 had a higher likelihood of recurrence compared to those with a lower LNR. These findings highlight the importance of considering LNR as a valuable prognostic marker for PTC patients with limited lymph node examination. Incorporating LNR into risk assessment can improve recurrence prediction and help guide treatment strategies. However, further prospective studies are needed to validate these results and determine their clinical implications in managing PTC patients.n = 675) and high-LNR (n = 234) groups was 0.29 . The recurrence rate was significantly higher in the high-LNR group compared to the rate in the low-LNR group . Multivariate analysis using Cox regression revealed that tumor size and LNR ≥ 0.29 were independent prognostic factors for recurrence. Therefore, LNR can be utilized to stratify the risk of recurrence in patients with low-LNY PTC.The American Thyroid Association risk stratification system suggests that having >5 metastatic lymph nodes (LNs) increase the recurrence risk in patients with papillary thyroid carcinoma (PTC). However, little is known about PTC with <5 harvested LNs. This study aimed to stratify patients with low-LN-yield (low-LNY) PTC based on lymph node ratios (LNRs). From 2007 to 2017, 6317 patients who underwent thyroidectomies were diagnosed with PTC at Seoul St. Mary’s Hospital, and 909 patients with low LNYs were included in the study. Tumor recurrence was compared based on LNR. The LNR cutoff was determined using a receiver operating characteristic curve. Forty-six patients (5.1%) experienced recurrences over a mean follow-up period of 127.24 ± 33.6 months . The cutoff for the low-LNR ( Thyroid cancer rates in the United States rose until the mid-2010s and then declined . In a reThe number of LNs surgically retrieved is referred to as the lymph node yield (LNY). Three factors affect LNY . SurgeonAccording to the American Thyroid Association (ATA) surgical affairs committee task force 2015 guidelines, ≤5 pN1 micrometastases of <0.2 cm in the largest dimension are classified as low-risk N1 disease with a recurrence rate of <5% ,8. In cop value 0.0184 vs. 0.3831) [The lymph node ratio (LNR) is defined as the number of metastatic LNs divided by the number of LNs collected. A systematic review of nine retrospective studies showed that LNR was an independent predictor of loco-regional recurrence in metastatic PTC . Further 0.3831) . The aimA total of 6317 patients who underwent thyroidectomies at Seoul St. Mary’s Hospital were diagnosed with PTC between January 2007 and December 2017. Among them, 909 patients with low LNYs were selected for inclusion in the study. The study included patients who underwent total thyroidectomy and central neck lymph node dissection (CLND), as our institution performed prophylactic CLND for all PTC patients during the study period. Patients with lateral neck metastasis or distant metastasis were not included in the study. The Institutional Review Board of Seoul St. Mary’s Hospital, The Catholic University of Korea, approved the study protocol (IRB No: KC23RISI0055). Informed consent was not required for this retrospective study and was waived by the committee.All patients included in the study were monitored in accordance with the management guidelines provided by ATA ,9. InitiWhen recurrent disease was suspected on routine follow-up examinations, additional diagnostic imaging studies, including neck computed tomography, were performed to determine the location and extent of the suspected recurrence. Recurrence was suspected when positive findings were noted on imaging studies or serum Tg levels increased significantly (≥50%). Recurrence was confirmed using ultrasound-guided fine-needle aspiration cytologies or surgical biopsies.t-tests were employed to compare continuous variables between the high- and low-LNR groups. Chi-squared or Fisher’s exact tests were used for the comparison of categorical variables. Univariate and multivariate Cox regression analyses were conducted to identify risk factors associated with recurrence. Receiver operating characteristic (ROC) curve analysis was employed to determine the optimal cutoff value for LNR in predicting recurrence, and sensitivity, specificity, and the area under the curve (AUC) with a 95% confidence interval (CI) were calculated. Statistical analyses were performed using IBM SPSS Statistics . Two-sided p values less than 0.05 were considered statistically significant.Mean and standard deviation were used to present continuous variables, while numbers and percentages were used for categorical variables. Student’s The baseline clinicopathological characteristics of the patients are shown in n = 234) and low-LNR group (n = 675). The cutoff value for LNR, determined by ROC curve analysis, was 0.29  as shown in p = 0.001), while the proportion of female patients was similar between the two groups . Tumor sizes were significantly larger in the high-LNR group compared to the low-LNR group . The high-LNR group had a higher percentage of patients with lymphatic invasion  compared to the low-LNR group. No significant differences were observed in terms of multifocality, ETE, vascular and perineural invasion, and BRAF mutations between the two groups. The high-LNR group had a significantly higher mean number of positive LNs compared to the low-LNR group , while the number of examined LNs was similar between the two groups . The rate of postoperative RAI ablation therapy was significantly higher in the high-LNR group compared to the low-LNR group . According to the eighth edition of the staging system by the American Joint Committee on Cancer/Union for International Cancer Control, the high-LNR group had a significantly higher TNM stage compared to the low-LNR group , while the T stage rate showed no significant difference (p = 0.129). About one out of ten patients in the low-LNR group developed LN metastasis (N1a). The recurrence rate was significantly higher in the high-LNR group compared to the low-LNR group .p < 0.001), positive LNs , LNR , minimal and gross ETE vs. no ETE , lymphatic invasion , LNR ≥ 0.29 , T2 and T3b vs. T1 , N1a , and stage II  were significantly associated with recurrence. Postoperative RAI was also a predictor for recurrence . Multivariate analysis revealed that tumor size and LNR > 0.29 were independent prognostic factors predicting recurrence . The Kaplan–Meier analysis demonstrated a significant difference in DFS between the high-LNR and low-LNR groups (log-rank p = 0.001)  .n = 547). A cutoff of LNR 0.29 predicted recurrence . Patients with high LNRs were more likely to be younger than patients with low LNRs . Lymphatic invasion occurred significantly more in the high-LNR group compared with the low-LNR group . The mean number of positive LNs for metastasis was significantly higher in the high-LNR group compared with the low-LNR group . All the patients in the high-LNR group were stage N1a, but only 60 out of 333 patients in the low-LNR group were stage N1a . The stage II rate was also significantly higher in the high-LNR group compared with the low-LNR group . Twenty-nine and seventeen patients had tumor recurrence in the high- and low-LNR groups, respectively .p < 0.001). The recurrence rate in the high-LNR group was significantly higher than the recurrence rate in the low-LNR group . In the subgroup analysis of patients who underwent postoperative RAI ablation, the high-LNR group developed more recurrence than the low-LNR group . Our results may help risk-stratify patients with PTC with a small number of harvested LNs, which was difficult under the current ATA risk stratification system.This study demonstrated that LNR can be used to stratify the risk of recurrence in patients with PTC with an LNY of five or fewer. LNR ≥ 0.29 was an independent prognostic factor for the risk of recurrence in patients with low-LNY PTC  patients experienced tumor recurrence during a follow-up of 127.24 ± 33.6 months. A meta-analysis that included six studies with 2939 patients surveyed between 2022 and 2013  demonstrated a recurrence rate of 4.4% in the total-thyroidectomy group (2134 patients) [We performed a subgroup analysis to determine if postoperative RAI therapy affected differences in the recurrence rates between the groups. Postoperative RAI ablation was once one of the main therapeutic strategies in patients with thyroid cancer but does not impact the recurrence rate, particularly in patients with low-risk thyroid cancer ,22,23. Ip < 0.0001) and lateral neck dissections  were associated with lower rates of recurrence in patients with PTC [p < 0.001) [Several groups reported data on the effects of LNY on prognosis in patients with PTC ,25,26,27p < 0.01) [Several studies focused on the relationship between LNR and the prognosis of patients with PTC, including the number and extent of metastatic LNs. Jeon et al. found that LNR > 0.4 was an independent risk factor for recurrence in pathological N1a PTC . Schneid < 0.01) . Pyo et  < 0.01) . Several < 0.01) . Zheng e < 0.01) . Weitzma < 0.01) . AlthougThere were several limitations due to the retrospective analysis. We included patients who underwent total thyroidectomy and RAI ablation therapy from 2007 to 2017. The ATA management guidelines were released in 2015; thus, the majority of patients were treated according to the former guidelines from 2009 [Nonetheless, our study has several strengths. We analyzed 909 patients and followed patients for 127.24 ± 33.6 months. To our best knowledge, our study is the first to verify the association between LNR and recurrence with long-term data, including patients with PTC who underwent surgery. The recent trend in PTC treatment is not to perform total thyroidectomy. Thus, future research should address the patients treated with thyroid lobectomies and unilateral central LN dissections.LNR ≥ 0.29 was an independent predictor of recurrence in patients with PTC with LNY ≤ 5. This finding may help follow-up patients with PTC after total thyroidectomy and cervical LN dissection. Further prospective studies are needed to substantiate our results.


HNTs and PLA-g-MA were added to the polymer blend at 5 and 10 wt.%, respectively, and everything was processed via melt compounding. A scanning electron microscopy (SEM) analysis shows that HNTs are preferentially localized in PHBHHx nodules rather than in the PLA matrix due to its higher wettability. When HNTs are combined with PLA-g-MA, a finer and a more homogeneous morphology is observed, resulting in a reduction in the size of PHBHHx nodules. The presence of HNTs in the polymer blend improves the impact strength from 12.7 to 20.9 kJ/mm2. Further, with the addition of PLA-g-MA to PLA/PHBHHX/HNT nanocomposites, the tensile strength, elongation at break, and impact strength all improve significantly, rising from roughly 42 MPa, 14.5%, and 20.9 kJ/mm2 to nearly 46 MPa, 18.2%, and 31.2 kJ/mm2, respectively. This is consistent with the data obtained via dynamic mechanical analysis (DMA). The thermal stability of the compatibilized blend reinforced with HNTs is also improved compared to the non-compatibilized one. Overall, this study highlights the effectiveness of combining HNTs and PLA-g-AM for the properties enhancement of PLA/PHBHHx blends.Given the global challenge of plastic pollution, the development of new bioplastics to replace conventional polymers has become a priority. It is therefore essential to achieve a balance in the performances of biopolymers in order to improve their commercial availability. In this topic, this study aims to investigate the morphology and properties of poly(lactic acid) (PLA)/ poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) (at a ratio of 75/25 ( The massive use of petroleum-based non-biodegradable polymers has led to serious environmental pollution due to the dissemination of plastics waste. To mitigate this propensity, scientists are committed to develop and synthesize biobased/biodegradable polymers with eco-friendly characteristics ,2. WithiPoly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) is characterized by a lower crystallinity and a wider processing window than the previous PHAs. Further, PHBHHx is soft with a high elongation at break and a relatively low tensile strength and modulus. Blending PHBHHx with PLA could be an effective way of improving the toughness and flexibility of PLA-based blends because of their complementary mechanical properties ,14. In tThe literature ,20,21 al2Si2O5(OH)4.H2O. They are used as fillers and reinforcements in polymers as a cheap alternative to expensive carbon nanotubes [g-MA compatibilizer revealed the occurrence of a synergistic effect between the compatibilizer and the nanofiller, which results in a significant increase in the thermal and mechanical properties of PHBV/PBS blends. From the short discussion above, the addition of nanofillers into a polymer blend matrix may act in two ways: first, they promote a transfer of stresses from the filler to the matrix, and secondly, they help to minimize phase separation and improve their interfacial adhesion. Although some papers on the compatibility of blends based on PLA/PHBHHx are reported in literature [g-MA as a compatibilizer on morphology and properties of a PLA/PHBHHx (75/25) blend prepared via melt compounding. The roles and performances of HNTs and PLA-g-MA in improving the morphology as well as the physical properties in terms of mechanical, thermal, viscoelastic, and flammability properties of the polymer blends are reported and commented.Among mineral fillers, halloysite nanotubes (HNTs) are another promising class of mineral clays with the molecular formula of Alanotubes . Indeed,anotubes . Moreoveanotubes . In thisanotubes  indicateanotubes  also obsanotubes . The autanotubes  performeterature ,40, to t−3; glass transition temperature (Tg) = 60 °C; and melting temperature (Tm) = 165 °C. PHBHHx in a pellet form was kindly supplied by Kaneka Corporation  as Aonilex X151 grade with a molar ratio of 3HHx = 10.5 mol. The polymer had the following physical characteristics: density = 1.19 gcm−3; Tg = −0.2 °C; and Tm = 130 °C. Halloysite nanotubes were supplied by the Algerian Kaolin Company  and were collected from the Djebel Debbagh mining area in northeastern Algeria (Guelma). Halloysite nanotubes (HNTs) have an average internal diameter of 10 to 30 nm, an external diameter of 30 to 50 nm, a length of approximately 0.1 to 2 microns, a density of 2.1 g cm−3, and a specific surface area of 51.4 m2/g [PLA in a pellet form was supplied by NatureWorks©  under IngeoTM, 2003D extrusion grade. The most important physical properties of the polymer are as follows: D-isomer content  = 4.3%; density = 1.24 gcm1.4 m2/g ,41. BefoReactive extrusion was used to graft maleic anhydride (MA) onto PLA chains. This method requires the optimization of various parameters including the content ratio of MA and DCP and the processing parameters, i.e., temperature and screw speed. According to the data in the literature ,43, the g-MA were dried in an oven overnight at 70 °C, while HNTs were dried at 105 °C for 12 h to prevent moisture uptake and any further polymer hydrolysis. Melt compounding of samples based on PLA, PHBHHx, and HNTs was carried out in a Thermo Haake Rheomix 600 internal mixer equipped with counter-rotating rotors operating at 50 rpm at 180 °C for 7 min. The formulation codes and compositions are given in g-MA was added at mid-processing time to ensure that the PLA melted. The resulting materials were ground and dried at 80 °C for 8 h. The next processing step was compression molding using the Model CARVER 3856 CE hydraulic press  to prepare standardized specimens for tensile and impact tests. For each form of characterization, the samples were put in a multi-adapter and covered with Teflon film heated to 180 °C between two metal plates. In detail, the polymeric materials were first pressed at low pressure for 2 min (3 degassing cycles), allowing for the material to melt and flow homogeneously. This was then followed by a high-pressure cycle at 10 bars for 4 min, and finally, the whole assembly was cooled to room temperature. Neat PLA and PHBHHx were subjected to the same experimental conditions.Prior to melt blending, PLA, PHBHHx, and PLA-g-MA was then dissolved in 20 mL of chloroform. The resulting solution was titrated with KOH (potassium hydroxide) (0.06M) in methanol in the presence of phenolphthalein as the color indicator. PLA-g-MA was totally soluble, and no precipitate was formed during titration.To determine the amount of maleic anhydride (MA) that reacted with PLA, the titration method was used ,45. FirsKOH is the volume of KOH; NKOH is the normality of KOH; 98.06 is the molecular weight of maleic anhydride; and WP is the tested weight.Three trials were conducted to ensure the reliability of the results. MA content was calculated using Equation (1) :(1)%MA%wg-MA. The FT-IR spectra were recorded in the 400–4000 cm−1 region with 32 scans at a resolution of 4 cm−1. This analysis was carried out directly on PLA and PLA-g-MA films.Grafting of maleic anhydride on PLA was studied by Fourier-transform infrared spectroscopy (FT-IR) using a Bruker spectrometer  in ATR mode to identify the functional groups of PLA and PLA-The fracture surface morphology of the samples was investigated using scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM/EDX) on a Quanta 250 FEI at a 10 kV acceleration voltage. The samples were cryogenically fractured in the transverse direction and coated with conductive carbon before analysis.Thermogravimetric analysis (TGA) measurements were performed on a Perkin Elmer STA 8000 thermal analyzer. Each sample at about 14 mg was subjected to a dynamic heating program from 30 to 600 °C at a heating rate of 10 °C/min under a nitrogen atmosphere. The reported values on the degradation temperatures represent an average of 3 tests.−1 at 23 °C. The values of Young’s modulus, elongation at break, and tensile strength represent an average of at least five measurements. Impact strength was determined using Izod impact tester  with an energy of 7.5 joules according to ASTM D256-10(2018) [Tensile measurements were performed using a Zwick/Z010 universal tensile testing machine . Dumbbell-shaped specimens (ISO 527-2:2012 type 1BA standards ) were te10(2018) . Notchedm represents the melting enthalpy obtained from the heating scan, ΔH°m is the melting enthalpy of a 100% crystalline PLA  experiments were performed using a Perkin-Elmer Diamond DSC . Samples of approximately 10 mg were packed in aluminum pans using a Perkin Elmer STA 8000 thermal analyzer. The samples were first heated from 30 to 200 °C at a heating rate of 10 °C/min. Next, the samples were cooled to 30 °C at a cooling rate of 10 °C/min; then, they were heated again to 200 °C at the same heating rate. The crystallinity index (Xc) of the samples is determined according to Equation (2) ,49:(2)Xc93.7 J/g , and PHB93.7 J/g  and w re2/O2 (80/20) atmosphere to carry out a complete combustion. The heat release rate (HRR) was calculated according to Huggett relation based on oxygen consumption (1 kg of oxygen consumed corresponds to 13.1 MJ of heat released) [To investigate flammability and combustibility, calorimetric measurements using pyrolysis-combustion flow calorimeter (PCFC) were carried out (Fire Testing Technology (FTT), East Grinstead, UK). A total of 2–4 mg of samples was subjected to an initial heating at 1 °C/s to 450 °C under a nitrogen atmosphere. Then, the pyrolysis products were directly transferred to a combustion chamber at 900 °C in a Neleased) .Viscoelastic properties of PLA, PHBHHx, and their blends were determined using a dynamic mechanical analyzer . The analysis was measured in tensile mode at a constant frequency of 1 Hz, and the displacement was fixed at 10 μm from −20 to 200 °C at 5 °C/min. The samples, in the form of bars with dimensions of 30 × 4 × 2 mm, were cut according to ISO 527-2 1BA dumbbells.g-MA recorded in transmittance mode in the domains of 4000–400 and 1830–1500 cm−1, respectively.FT-IR spectroscopy was used to observe any changes occurring in the chemical structure of PLA after the grafting reaction with MA in the presence of DCP, which was used as the radical initiator. max = 1767 cm−1 of a relatively high intensity, while the other one of a less intensity appears at λmax = 1850 cm−1. These two absorption bands are due to the symmetrical and asymmetrical stretching of carbonyl groups (C=O), respectively. Typical absorption bands in the FT-IR spectrum of the clean PLA are clearly visible at 1780–1700, 1500–1320, and 1300–1000 cm−1, and are correlated with C=O stretching, CH and CH3 bending, and C-O stretching, respectively. This is consistent with the data found in the literature [In terature .g-MA shows a broadening absorption band in the range of 1886-1850 cm−1, which is not observed for neat PLA, as illustrated in g-MA at about 1630 and 1562 cm−1, which are not observed for neat PLA, as clearly shown in g-MA, suggesting that the MA molecules are grafted onto PLA backbones, converting MA into saturated anhydride (succinic anhydride).The FT-IR spectrum of PLA-The amount of grafted MA is determined with the titration method using Equation (1), and the value obtained is approximately 0.62% MA unit grafted onto PLA. The functionalization of PLA should result in high reactivity with a wide range of polymer chains and biological macromolecules .g-MA compatibilizer, either separately or combined, on the microstructure of the PLA/PHBHHx (75/25) blend. Accordingly, the SEM micrographs of the fracture surface of the PLA/PHBHHx blend with 5 wt.% of halloysite nanotubes with and without 10 wt.% of PLA-g-MA are shown in A morphological investigation is of crucial importance to evaluate the effect of HNTs and PLA-g-MA, respectively. In g-MA to the PLA/PHBHHx/HNT ternary blend provides a good dispersion of HNT particles in the polymer matrix, as we also observe the disappearance of micronic aggregates of the mineral filler. The SEM micrographs clearly show that the addition of PLA-g-MA to the PLA/PHBHHx/HNT nanocomposite significantly improves the morphology of the ternary blend. The size of the nodules is drastically reduced, and the fracture surface appears to be less sharp, indicating a more homogeneous morphology than the non-compatibilized one.According to the literature ,25, the g-MA compatibilizer is examined more closely at a higher magnification, i.e., 10,000×, as shown in For this purpose, the position of halloysite nanotubes (HNTs) in the PLA/PHBHHx blend in the absence and presence of a PLA-g-MA to the blend  blend is characterized by intermediate tensile properties between stiffness and ductility. This behavior has already been reported by Zhao et al. [o et al.  and Lim o et al.  in theirAs reported in the literature ,58, whenIndeed, the hydrophilic nature of halloysite nanotubes prevents any compatibility with the highly hydrophobic polymer blends. In addition, the SEM images indicate a relatively low content of HNTs in the PLA phase. This non-uniform distribution of HNTs in the nanocomposite may weaken the interfacial adhesion and lead to a reduction in the tensile strength . Interesg-MA to improve the strain resistance of the PLA/PHBHHx blend. Indeed, the percentage increases in the tensile strength, elongation at break, and impact strength are approximately 12, 25, and 50% compared with the blend filled with halloysite nanotubes, which is still much higher than the unfilled PLA/PHBHHx blend. The data in g-MA with the hydroxyl groups of HNTs and PHBHHx chains may be responsible for promoting the interfacial adhesion between them, which is in good agreement with the morphological observations  is almost 285 °C and falls between that of PLA, which is roughly 335 °C, and PHBHHx, which is about 256 °C. The increase in T5 may be due to a good dispersion of halloysite nanotubes in the polymer blend. Moreover, according to some authors [g-MA. The T5 value of the virgin PLA/PHBHHx blend increased from 285 °C to about 290 and 291 °C in the presence of only halloysite nanotubes and in the presence of both HNTs and PLA-g-MA, respectively.From  authors , the tub authors . The theHowever, for the second stage of degradation (more than 27% mass loss), the curve shifted to lower temperatures. This unexpected observation can be attributed to a hydrolysis phenomenon, resulting from several factors including the dispersion state of HNTs in the PLA matrix, and agglomerated halloysite nanotubes, which are also able to create localized regions in a higher water concentration, speeding up hydrolysis in these zones. It is also plausible that the products resulting from the first degradation of PHBHHx plays an active role in the PLA degradation process. Polymeric degradation often leads to the formation of low-molecular-weight compounds, such as oligomers, monomers, and smaller fragments. These degradation products may possess functional groups or chemical motifs that are sensitive to hydrolysis. On the other hand, Prasad et al.  and Yuan1 and Tmax2), which correspond to PHBHHx and PLA, respectively. The Tmax of PLA in the blend shifts to lower temperatures, whereas that of PHBHHx (Tmax2) slightly increases. For the nanocomposite reinforced with halloysite nanotubes, Tmax1 slightly increases, while Tmax2 decreases. For the nanocomposite compatibilized with PLA-g-MA, there is a slight increase in the Tmax1 (+5 °C) and a significant increase in the Tmax2 (+11 °C) compared with the Tmax of the nanocomposite before compatibilization.g-MA may be due to an improvement in the PLA-HNT interfacial adhesion. The hydroxyl groups of HNTs likely react with MA groups to form covalent bonds between the filler and PLA. This reaction reduces the reactivity of hydroxyl groups for other chemical reactions, including hydrolysis mechanisms.An improvement in the Tmax of PLA in the presence of PLA-In order to estimate the possible interactions between the components of the blend, the mass loss value was calculated from the separate data for each component as the total of the individual contributions, assuming that there were no interactions. The theoretical TGA curve for the case of a binary blend was built using Equation (3) according to ,66. Thiseference , when thAs shown in g-MA shows a significant variation in the crystallization and melting temperature of PLA. It is observed in A comparison between the DSC thermograms of neat PLA/PHBHHx and those of the nanocomposite with PLA-The presence of double/multiple melting peaks is a frequently observed phenomenon in semi-crystalline aliphatic polyesters, which is due to the presence of melting and recrystallization processes. During heating, PLA with imperfect crystals melts first, followed by the remelting of PLA with more perfect crystals . This phPCFC was used to assess the sample’s flammability behavior. The temperature-dependent heat curves are shown in 2 content in the chemical structure of PHBHHx are higher than those of PLA. This chemical composition may contribute to the greater combustibility of PHBHHx compared with PLA. Two degradation phases are detected in the flammability behavior of the polymers, similar to what the TGA reported. The heat release rate (HRR) curves of the PLA/PHBHHx and PLA/PHBHHx/HNT blends reveal two unique peaks, which correspond to PHBHHx and PLA flammability. The presence of halloysite nanotubes (HNTs) reduces the temperature heat release rate (TpHRR) of PHBHHx and PLA compared to the neat blend. The TpHRR of PHBHHx shows a slight decrease, while the TpHRR of PLA decreases significantly by around 22 °C. This result suggests the occurrence of interactions between HNTs and the polymers, particularly PLA, since the incorporation of HNTs seems to promote hydrolysis and thermal decomposition reactions, leading to a faster release of flammable compounds. This is in a good agreement with the TGA results.The curves of the peak heat release rate (pHRR) of the neat PHBHHx and neat PLA show that the two polymers have different combustion behaviors. The total heat release (THR) values for PLA and PHBHHx are 16.5 and 21.9 kJ/g, respectively, indicating that PHBHHx releases more heat during combustion than PLA. The difference in THR can be attributed to several factors such as differences in the chemical composition, molecular structure, and thermal properties of the polymers. For example, the C/O ratio and -CHg-MA results in a slight increase in the TpHRR for both PLA and PHBHHx in the nanocomposite blend, suggesting a beneficial effect of PLA-g-MA on the thermal behavior of the blend.The incorporation of PLA-g-MA nanocomposite. The storage modulus (E’) and tan δ curves are presented in g-MA) follows a multi-step chain relaxation process. At a temperature below the glass transition of PHBHHx, the E’ exhibits a relatively stable plateau, although a slight disturbance in the E’ values is observed. As the temperature increases, the E’ of PLA gradually decreases. g-MA to the blend results in a higher storage modulus (E’) compared to the blend over the entire temperature range up to the transition temperature of PLA. This shows the reinforcing effect of halloysite nanotubes and the enhanced dispersion of HNTs in the blend matrix due to the compatibilizer.DMA was performed on the samples based on the PLA/PHBHHx blend, PLA/PHBHHx/HNT, and the PLA/PHBHHx/HNTs/PLA-g-MA nanocomposites is displayed in g-MA nanocomposites compared with that of the binary PLA/PHBHHx (75/25) blend. g-MA has no significant effect on tan (δ).The temperature dependence of the loss moduli (E″) of the PLA/PHBHHx blend, PLA/PHBHHx/HNT, and the PLA/PHBHHx/HNTs/PLA-g-MA suggests that no substantial cross-linking or grafting reaction has occurred between PLA and PHBHHx. It should be noted that the absence of significant changes in the presence of PLA-g-MA does not necessarily negate the overall improvement in the mechanical properties or compatibility achieved by other means. The effectiveness of PLA-g-MA as a compatibilizer and the reinforcing effect of halloysite nanotubes may contribute to the overall improvement in the mechanical performance of the nanocomposite.The absence of significant differences in the results of the dynamic mechanical analysis with the presence and absence of PLA-g-MA) produced via reactive extrusion was used. The FT-IR spectra and chemical titration confirmed the grafting of MAs onto the PLA matrix, and the amount of grafted MA reached 0.62 wt.%. Morphological, thermal, and mechanical characterizations were performed to understand the influences of HNTs and PLA-g-MA on PLA/PHBHHx (75/25 wt%).Polymer blends reinforced with natural fillers are very important for developing new materials with enhanced properties, especially when these materials are bio-based and totally biodegradable. This research investigates the structure–property relationships of the novel ternary poly(lactic acid) (PLA)/poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx)/halloysite nanotubes (HNTs) nanocomposites prepared via melt blending. In order to achieve a better interfacial interaction between the polymer components, a laboratory-synthesized compatibilizer of maleic anhydride-grafted PLA (PLA-g-MA compatibilizer into PLA/PHBHHx/HNT, there is an increase in the tensile strength, elongation at break, and impact resistance by about 12, 25, and 50%, respectively, compared to the non-compatibilized ones. Further, a slight increase in the thermal stability of the compatibilized nanocomposite is also observed.From this study, the following conclusions can be drawn: The SEM observations show that the halloysite nanotubes are preferentially located in the PHBHHx nodules due to a better HNT-PHBHHx wettability. The combination of HNTs and a compatibilizer results in a homogeneous morphology with a decrease in the size of the PHBHHx nodules. The incorporation of HNTs into the PLA/PHBHHx blend results in a slight decrease in the tensile strength of the nanocomposites, while the modulus and elongation at break remain almost unchanged. However, the impact resistance increases by 22% compared to that of the neat PLA/PHBHHx blend. Furthermore, after the addition of a PLA-g-MA is an additional approach to improve the performance properties of PLA/PHBHHx blends.This article shows that blending biopolymers is an effective and promising way to adjust some of their properties. So, the use of halloysite nanotubes with a suitable compatibilizer such as PLA-


Porphyromonas gingivalis (Pg), the keystone pathogen of the oral cavity, can induce a systemic antibody response measured in patients’ sera using enzyme-linked immunosorbent assays. The present case–control study quantified the immune system’s response to Pg abundance in the oral cavities of patients affected by different central nervous system pathologies. The study cohort included 87 participants: 23 healthy controls (HC), 17 patients with an acute neurological condition (N-AC), 19 patients with a chronic neurological condition (N-CH), and 28 patients with neurodegenerative disease (N-DEG). The results showed that the Pg abundance in the oral cavity was higher in the N-DEG patients than in the HC (p = 0.0001) and N-AC patients (p = 0.01). In addition, the Pg abundance was higher in the N-CH patients than the HCs (p = 0.005). Only the N-CH patients had more serum anti-Pg antibodies than the HC (p = 0.012). The inadequate response of the immune system of the N-DEG group in producing anti-Pg antibodies was also clearly indicated by an analysis of the ratio between the anti-Pg antibodies quantity and the Pg abundance. Indeed, this ratio was significantly lower between the N-DEG group than all other groups . The immune system’s response to Pg abundance in the oral cavity showed a stepwise model: the response diminished progressively from the patients affected with an acute condition to the patients suffering from chronic nervous system disorders and finally to the patients affected by neurodegenerative diseases. Previous studies have reported an association between oral microbial dysbiosis and the development and progression of pathologies in the central nervous system.  The oral cavity is characterized by distinctive features, making it an ideal habitat for a collection of microorganisms . The mouPorphyromonas gingivalis (Pg) is a Gram-negative, nonmotile short rod, and an anaerobic bacterium, considered to be the keystone pathogen of the oral cavity because it is implicated in the biofilm formation of bacterial plaques. With other oral microbes, it forms a biofilm that alters tight epithelial junctions and promotes inflammation by mucosal immune cells. Pg can modulate the host immune response, at first increasing nutrient availability and biofilm growth, and subsequently facilitating bacterial resistance by destroying complement factors [Pg produces many virulence factors, such as proteases, endotoxins, organic acids, and key enzymes, including the proteases known as gingipains [Pg inhibits the conversion of M2 macrophages by redirecting them to the M1 inflammatory subtype, worsening the inflammatory environment. M1 may exhibit cytotoxic and M2 neuroprotective effects [Pg promotes the onset of chronic periodontal disease, which is characterized by a systemic antibody response, measured by the serum levels of antibodies against periodontal pathogens [ factors . Pg prodngipains . Gingipangipains . Pg inhi effects . Chronicathogens ,8.Pg were detected in subjects before cognitive impairment [Pg antibodies were also detected in neurological patients’ sera using enzyme-linked immunosorbent assays (ELISA) [The immune system preserves the microenvironment for the microbiota maintaining local and systemic homeostasis and preserving host biological integrity . The prepairment ,16. Anti (ELISA) .Pg-induced inflammation in the oral cavity can be transferred to the CNS. Pg lipopolysaccharide (LPS) can reach the brain-resident microglia via the leptomeningeal cells, which express Pg-LPS receptors (TLR2 and TLR4). Liu et al. demonstrated that these cells can transmit inflammatory signals from peripheral macrophages to the brain-resident microglia due to Pg-LPS stimulation [Pg has been detected in AD patients’ brain tissues compared to healthy volunteers [Pg titers and neuroinflammation processes [Specific oral bacterial species have also been implicated in several systemic diseases, such as bacterial endocarditis , aspiratmulation . Furtherlunteers . AD incirocesses .Recently, a novel pathogenic model, brain-first versus body-first, was proposed to classify two different subtypes of PD. According to this model, in the brain-first (top-down) type, α-synuclein pathology could initially arise in the brain with secondary spreading to the peripheral autonomic nervous system. In the body-first (bottom-up) type, the pathology could originate in the enteric or peripheral autonomic nervous system and then spread to the brain. However, these findings are still under further investigation .Proactive approaches to balancing oral dysbiosis include the use of probiotics, paraprobiotics, postbiotics, and ozone therapy .Pg abundance in the oral cavities of patients affected by pathologies in the CNS. In particular, anti-Pg antibodies in the serum and the Pg abundance in the oral cavity were quantified in patients with neurodegenerative diseases compared to healthy controls (HC) and patients affected by acute or chronic neurological diseases. In this study, we evaluated the immune system’s response to The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the local Ethics Committee of the “G. d’Annunzio” University of Chieti-Pescara on May 2020 (ethics code: 1909).p = 0.037, with a statistical power of 60.4% considering the probability of type I error α = 0.05) between 15 patients affected by neurodegenerative diseases and 21 patients affected by neurological non-neurodegenerative diseases in the ratio between anti-Pg antibodies’ quantity and Pg abundance. In the same condition, the statistical power could reach 80% with α = 0.05 if a sample size of 33 was considered for each group. In addition, also considering the HC group, we estimated that, with a low percentage increase of 2.4% in the ratio values, we could obtain a statistical power of 80% and α = 0.05 with a sample size of 30 for each group. Accordingly, the eligibility criteria were evaluated in 30 HC and 90 neurological patients, who could be subdivided into three groups, obtaining 1:1 matching among the groups. HCs free from neurological diseases were recruited from a list of HCs. In contrast, from May 2020 to July 2022, all patients were enrolled in the Neurology Clinic of “SS Annunziata” Hospital of Chieti. Individuals under antibiotic therapy (2 HCs and 8 patients) or those using daily chlorhexidine mouthwash within the last 3 months (3 HCs and 7 patients) were excluded from the study. In addition, 5 patients were excluded for concomitant tumors, and 2 HCs and 6 patients declined to participate. Thus, the final cohort included 23 HCs, 17 patients with an acute neurological condition (N-AC), 19 patients with a chronic neurological condition (N-CH), and 28 patients with a neurodegenerative disease (N-DEG). Written informed consent was obtained from all 87 participants. For each individual, tongue biofilm was used to quantify the Pg abundance and a blood sample was used to quantify the anti-Pg antibody levels.A previous pilot study  found a n = 7), head trauma (n = 6), encephalitis (n = 2), loss of consciousness (n = 1), and cerebrospinal fluid hypotension (n = 1).The N-AC group included patients referred to the Neurology Clinic for the following reasons: ischemic stroke (n = 7), myasthenia (n = 6), polyneuropathy (n = 4), brain tumor (n = 1), and vascular leukoencephalopathy (n = 1).The N-CH group included patients with a diagnosis of a chronic neurological condition such as: epilepsy , multiple sclerosis (n = 6), sclerosis lateral amyotrophic (n = 4), mixed dementia (n = 3), Alzheimer’s disease (n = 2), Frontotemporal dementia (n = 2), and Huntington’s disease (n = 1). The N-DEG group included patients with the following diagnoses: Parkinson’s disease  and then kept at +4 °C until the nucleic acid extraction. A blood sample was also taken from all the participants on the same day of the brushing and stored at −80 °C.Oral examinations were performed by a trained dentist (P.P.) using mirrors , a dental probe , and an intra-oral light. The teeth number, plaque index (PI), and gingival index (GI) were recorded using the standardized Oral Health Questionnaire . All othPg ATCC 33277  was used and cultivated as previously reported [12 CFU. Molecular analyses were performed to quantify the Pg in the oral cavities of all the participants. The total genomic DNA was isolated from the samples and the reference bacterial strain using a Quick DNA miniPrep Plus KIT . StepOne™ 2.0  was used in a qPCR analysis to quantify the Pg abundance in each sample. A TaqMan-based assay that recognizes Pg 16S rRNA, the gene encoding the small subunit of 16S ribosomal RNA, was used, as previously reported [Pg 16S rRNA gene. This method allowed us to estimate the bacterial quantity based on the amount of total DNA isolated from the oral samples [In this study, the reference bacterial strain reported . Brieflyreported . A stand samples .Pg using the ChonBlockTM buffer system  as follows [TM standard/sample dilution buffer. Moreover, Blocking Buffer was added to each well and incubated for 1 h; the plate was washed using Wash Buffer; the standards and samples were loaded and incubated for 2 h; the plate was washed using Wash Buffer; secondary antibody solution was loaded and incubated for 1 h; the plate was washed using Wash Buffer; TMB solution was added and incubated for 25 min; and finally, Stop Solution was added and the OD values at 450 nm were recorded within 5 min. The antibody levels were determined by comparing them to standard levels and were analyzed using a regression analysis. The antibody levels were expressed as EU (103 units/mL).The ELISA technique assayed the serum IgG antibody responses to the bacterial pathogens from  follows . SpecifiPg abundance and anti-Pg antibody quantity. The level of significance for all the statistical analyses was set at 0.05.The data were compared among the groups using non-parametric Kruskal–Wallis tests. Levene’s test was used to test the homogeneity of the variance among the groups. A post hoc test was applied for pairwise comparisons. Spearman’s correlation tests were performed on all the participants to evaluate the associations between the variables. Spearman’s correlation tests were also performed separately for each group to evaluate the effect of age and sex on the The demographic and oral characteristics of the study cohort are shown in 2(3) = 10.4, p = 0.02). The post hoc comparisons showed that only the N-AC group was significantly older than HCs (p = 0.02). Sex, smoker status (%), and former smoker status (%) were not significantly different among the groups (χ2(3) = 4.4, p = 0.22, χ2(3) = 1.3, p = 0.73, and χ2(3) = 4.6, p = 0.21, respectively). The teeth number significantly differed among the groups (χ2(3) = 13.9, p = 0.003). The post hoc comparisons showed that HC group had a higher teeth number than the N-AC (p = 0.008), N-CH (p = 0.04), and N-DEG (p = 0.02) groups. No significant differences among the groups were found in the number of fixed prostheses (p = 0.27) and removable dentures (p = 0.17). Due to the low number of participants who completed the dental visit, statistical comparisons among the groups were not performed on other oral indices.Age significantly differed among the groups . A significant correlation was also found between the Pg abundance, age , and sex . These correlations are shown in Pg in the oral cavity increased with age and in males. A significant correlation between age and Pg abundance was found for the HC group only . Indeed, the N-AC, N-CH, and N-DEG groups showed no significant correlations between Pg abundance and age . No significant correlation was found between Pg abundance and sex at the group level . No significant correlation was found between the abundance of Pg and smoking (p = 0.35), prior smoking (p = 0.99), teeth number (p = 0.44), number of fixed prostheses (p = 0.54), and number of removable dentures (p = 0.22). No significant correlation was found between anti-Pg antibodies’ quantity and age (p = 0.90), sex (p = 0.07), smoking (p = 0.54), or former smoking (p = 0.53). Spearman’s correlation showed a significant positive correlation between the abundance of Pg bacteria and antibody quantities among the groups. Specifically, logarithmic transformations were applied to the Pg abundance value, anti-Pg antibodies’ quantity, and the ratio between anti-Pg and Pg to respect the homogeneity of the variance among the groups, as assessed by Levene’s test. The Kruskal–Wallis H test showed that the log abundance of Pg in the oral cavity was significantly different among the groups (χ2(3) = 31.9, p = 0.0001), with a mean rank value of 23.1 for the HC, 38.2 for the N-AC, 48.8 for the N-CH, and 61.4 for the N-DEG groups. The post hoc test showed that the Pg abundance in the oral cavity was higher in the N-DEG group than the HC (p = 0.0001) and N-AC (p = 0.01) groups, and it was also higher in the N-CH group than the HCs (p = 0.005).Non-parametric Kruskal–Wallis tests were performed to compare the Pg antibodies was significantly different among the groups (χ2(3) = 10.0, p = 0.020), with mean rank values of 31.8, 42.8, 55.9, and 46.7 for the HC, N-AC, N-CH, and N-DEG groups, respectively. The post hoc test showed that the anti-Pg antibodies in the serum were higher in the N-CH group than the HCs (p = 0.012).The log of anti-Pg antibodies’ quantity and the Pg abundance was significantly different among the groups (χ2(3) = 37.5, p = 0.0001), with mean rank values of 65.4 for the HC, 50.4 for the N-AC, 43.8 for the N-CH, and 22.7 for the N-DEG groups. The post hoc test showed that the log of the ratio between the anti-Pg antibodies’ quantity and the Pg abundance was lower in the N-DEG group than the HC (p = 0.0001), N-AC (p = 0.002), and N-CH (p = 0.03) groups, and it was also lower in the N-CH group than the HCs (p = 0.04). The log of the ratio between the anti-Pg abundance logarithm, the anti-Pg antibodies’ logarithm, and the logarithm of the ratio between the anti-Pg antibodies and Pg abundance for each group. Pg abundance in the oral cavity was higher in people with chronic neurological conditions than in the HCs or people affected by an acute neurological condition. Previous studies have shown that the oral microbiota composition originating from buccal and sublingual mucosa and salivary samples differs between healthy subjects and PD patients [This study showed that the patients ,46,47,48patients ,50. Unlipatients .Pg and Treponema denticola, are increased in subjects with cognitive impairment or AD [Pg colonization, and commensal-turned pathobionts could contribute to inflammation [Pg cysteine proteases. A Pg increase in neurological patients may lead to a higher delivery of gingipains, which are involved in the degradation of the tight junction proteins of cerebral microvascular endothelial cells , promoting damage to the blood–brain barrier [Pg produces two forms of gingipains: the free form and the outer membrane vesicles (OMVs)-associated form. Probably, via the OMVs, gingipains may be transported and released, increasing the permeability of human cerebral microvascular endothelial cells [Periodontal pathogens, such as nt or AD ,53. The ammation . The mec barrier . Pg prodal cells .Pg abundance was also increased with age, confirming previous findings [Pg abundance and age was significant for the HCs only. This result suggests that neurological conditions bother the relationship between age and the abundance of Pg bacteria in the oral cavity. The sex effect could have been related to the greater number of males in the N-DEG group, which could have biased the result toward an increased abundance of Pg in males compared to females. Indeed, when the correlation analyses were performed on single groups, the correlation between Pg abundance and sex was lost.In our study, findings . HoweverPg in the oral cavity and the quantity of anti-Pg antibodies in the serum of all the participants, confirming the hypothesis that oral bacteria can cause the production of anti-Pg antibodies, as previously reported [A significant positive correlation was found between the abundance of reported ,57. In areported .Pg in the N-DEG group, the quantity of anti-Pg antibodies was not higher in this group than the others. The inadequate response of the immune system of the N-DEG group in producing anti-Pg antibodies was revealed by the analysis of the ratio between the anti-Pg antibodies’ quantity and the Pg abundance. Thus, a low level of antibodies could result in a chronic inflammatory state inside and outside the oral cavity, which might be involved in the processes of neurodegeneration. Elevated IgG antibodies against periodontal disease bacteria have been found in subjects years before cognitive impairment, whereas IgG against Pg was decreased in patients who developed AD [Pg can regulate immune responses by creating an inflammatory environment through the stimulation of TLR2-PI3K-mediated signaling, which results in a reduction in bactericidal activity, an increase in proinflammatory cytokines , and the inhibition of phagosome formation and maturation [Interestingly, despite the higher amount of loped AD . Chronicloped AD ,60. Therturation .Pg antibodies than the HCs. However, the amount of these antibodies was inadequate compared to the HCs, as indicated by the significant difference between the N-CH and HC groups in the ratio between the anti-Pg antibodies and Pg. However, this ratio was higher in the N-CH than in the N-DEG group, suggesting that the low production of anti-Pg antibodies with a high abundance of Pg in the oral cavity was related more to neurodegeneration than to chronic neurological disease. On the contrary, the patients affected by an acute neurological condition  included in the N-AC group showed no significant difference compared to the HC group. Indeed, the HC and N-AC groups showed higher antibodies in the presence of a low abundance of Pg in the oral cavity.The N-CH group showed a higher quantity of anti-The accumulation of bacterial plaques by periodontopathogen bacteria, in addition to cigarette smoking, is the most important risk factor in causing an inflammatory response ,63. To rLactobacillus reuteri, which can reduce the amount of periodontopathogens and proinflammatory cytokines [Other therapeutic approaches include prebiotics, probiotics, paraprobiotics, and post-biotics. An example of probiotic therapy is ytokines ,67. All ytokines ,69,70.The main limitation of our study is its relatively small sample size and the heterogeneity of the patient groups. Unfortunately, a considerable amount of participants were excluded or dropped out and the recruitment could not continue. Thus, we could not draw definitive conclusions about this oral bacterium’s influence and the altered immune response in the pathogenesis of neurodegenerative diseases. Future follow-up studies could monitor the microbiota profile and the immune response over time and at different stages of disease to investigate the influence of periodontopathogen bacteria on the onset and progression of neurological and neurodegenerative diseases. Pg abundance in the oral cavity, assessed by the quantification of anti-Pg antibodies in the serum, showed a stepwise model. The immune system response decreased progressively in patients affected by an acute condition such as ischemic stroke or head trauma, less in patients with epilepsy or peripheral nervous system disorders, and finally, even less in patients affected by neurodegenerative diseases such as Parkinson’s or multiple sclerosis. In conclusion, the response of the immune system to


Background: Substance use continues to be on the rise in the United States and has been linked to new onset cardiovascular diseases (CVDs) and cerebrovascular disorders (CeVDs). We aimed to study the association between the types of substance use disorders (SUDs) with specific subtypes of CVDs and CeVDs among hospitalized patients using the National Inpatient Sample (NIS) Database.Methods: A retrospective study of the NIS database (2016-2017) using the ICD-10-CM codes was performed. The hospitalizations with a secondary diagnosis of SUDs were identified. Weighted univariate analysis using the Chi-square test and multivariate survey logistic regression analysis was performed to evaluate for the incidence, prevalence, and odds of association between vascular events and SUDs.Results: There were a total of 58,259,589 hospitalizations, out of which 21.42% had SUDs. SUDs were more common in the younger age group of 18-50, males, and the lower median household income group. We found a significant association of acute ischemic stroke (AIS) with amphetamine dependence , cocaine-related disorders , and nicotine dependence . There was a significant association between intracerebral hemorrhage with amphetamine dependence , cocaine-related disorders , and alcohol-related disorders . The association of subarachnoid hemorrhage (SAH) was noted to be higher with amphetamine dependence  and nicotine dependence . The patients with nicotine dependence had greater odds of having a myocardial infarction , those with cocaine-related disorders had higher odds of having angina pectoris , and patients with alcohol-related disorders had higher odds of developing atrial fibrillation  in comparison to non-SUDs.Conclusion: Our study demonstrates the variability of CVD and CeVD in patients hospitalized for SUD. Findings from our study may help promote increased awareness and early management of these events. Further studies are needed to evaluate the specific effects of frequency and dose on the incidence and prevalence of CVD and CeVD in patients with SUD. The past few decades have seen a rise in the incidence and prevalence of cardiovascular disease (CVD) concomitant with substance use disorders (SUDs) among younger adults aged 18-45 years of age .The SUDs comprise an estimated 6.7% of all inpatient admissions and has been associated with greater costs and longer hospitalizations relative to non-SUD admissions . A recenHowever, there is known variability in the types of CV events, depending on the type of agent used. For instance, an analysis of the National Health and Nutrition Examination Survey (NHANES) revealed a statistically significant association of non-fatal myocardial infarction with lifetime users of cocaine, when compared with non-users. The study did not reveal a significant association between ischemic stroke with cocaine users . We, theDetails of dataus.ahrq.gov/db/nation/nis/nisdde.jsp.Data were obtained from the Agency for Healthcare Research and Quality's Healthcare Cost and Utilization Project (HCUP) Nationwide Inpatient Sample (NIS) files between January 2016 and December 2017. The NIS is the largest publicly available all-payer inpatient care database in the United States and contains discharge-level data provided by states that participate in the HCUP . This administrative dataset contains data on approximately 8 million hospitalizations in 1,000 hospitals that were chosen to approximate a 20% stratified sample of all US community hospitals, representing more than 95% of the national population. Criteria used for stratified sampling of hospitals into the NIS include hospital ownership, patient volume, teaching status, urban or rural location, and geographic region. Discharge weights are provided for each patient discharge record, which allows extrapolation to obtain national estimates. Each hospitalization is treated as an individual entry in the database and is coded with one principal diagnosis, up to 29 secondary diagnoses, and 15 procedural diagnoses associated with that stay. Detailed information on NIS is available at http://www.hcup-Demographic characteristics of the populationhttps://www.icd10data.com/ICD10CM/Codes/F01-F99/F10-F19. The SUDs include alcohol-related disorders, amphetamine dependence, cannabis-related disorders, cocaine-related disorders, hallucinogen-related disorders, inhalant-related disorders, opioid-related disorders, and nicotine dependence.We performed a retrospective cross-sectional observational study on adult hospitalizations in the USA from 2016 to 2017. Secondary diagnoses of SUDs were identified using the ICD-10-CM code. Here are the listed ICD codes for SUDs that we used in our study, The comparison was made between US hospitalizations with a history of SUDs and hospitalizations without a history of SUDs. In each group, primary and secondary diagnoses of CVDs and CeVDs were identified. We considered angina pectoris, myocardial infarction (STEMI), sudden cardiac arrest, ischemic heart disease, and atrial fibrillation as CVDs and acute ischemic stroke (AIS), transient ischemic attack, and hemorrhagic stroke as CeVDs. ICD-10-CM codes for vascular diseases are described in Table OutcomesThe primary aim of this study was to evaluate the incidence and prevalence of CVDs and CeVDs amongst patients with SUDs. The secondary aim of this study was to evaluate the odds of CVDs and CeVDs amongst SUDs.Statistical analysist-test was used for continuous variables. Multivariable survey logistic regression models were generated to predict the association of CVDs and SUDs as well as CeVDs and SUDs after adjusting for confounding variables. There was no pre-decided sample size calculation. The p-value <0.05 was considered significant and OR with its 95% CI was calculated.We analyzed the data using SAS software (Version 9.4) . We performed univariate analysis to find the association of SUDs with CVDs and CeVDs. Categorical variables were evaluated using Chi-square and a Details of confoundersIn regression analysis, the models were adjusted with socio-demographic variables like age, race, ethnicity, annual household income, education status, and comorbidities like diabetes, cholesterol, hypertension cardiovascular health, obesity, and preventive aspirin use.Disease hospitalizationsWe found 58,259,589 hospitalizations  from January 2016 to December 2017 after excluding patients with ages <18 years. Out of these hospitalizations, 21.42% had a history (secondary diagnosis) of major SUDs. The prevalence of alcohol-related disorders was 5.87%, amphetamine dependence was 0.51%, cannabis-related disorders was 1.84%, cocaine-related disorders were 0.59%, hallucinogen-related disorders were 0.01%, opioid-related disorders were 1.53%, and nicotine dependence was 11.07%.Demographic characteristics of patients with a history of substance abuseAmong patients admitted to the hospital with a diagnosis of SUD, the mean age was found to be relatively lower for all the subtypes of SUDs: alcohol-related disorders , amphetamine dependence , cannabis-related disorder , cocaine-related disorders , hallucinogen-related disorders , inhalant-related disorders , opioid-related disorders , and nicotine dependence  in comparison to patients without a diagnosis of SUD  (p < 0.0001). The patients were divided into three age groups for subgroup analysis: 18-50, 50-75, and >75 years old. All SUDs were more prevalent in the age group of 18-50 in comparison to the other groups. Males had a higher prevalence of all types of SUDs in comparison to females (p < 0.0001). Among all hospitalized patients, Native Americans were found to have a significantly higher prevalence of alcohol-related disorders, amphetamine dependence, inhalant-related disorders, opioid-related disorders, and nicotine dependence in comparison to other ethnic groups (p < 0.0001). Similarly, African Americans had a significantly higher prevalence of cannabis-related disorders, cocaine-related disorders, and hallucinogen-related disorders (p < 0.0001). We performed a subgroup analysis to look for differences in the prevalence of SUDs among hospitalized patients belonging to different economic backgrounds. The patients with the lowest median household income (0-25th percentile) had a higher prevalence of all types of SUDs in comparison to other household income groups (p < 0.0001). The admitted patients who were self-payers had a significantly higher prevalence of alcohol use disorders and nicotine dependence as compared to all other insurance groups . The rest of the SUDs was more prevalent among the patients insured with Medicaid (p < 0.0001). Among all hospitalized patients with a diagnosis of SUD, a significantly higher proportion of patients were admitted over the weekend (p < 0.0001) , and nicotine dependence 2.28%) when compared to those without SUDs (1.70%) (p < 0.0001). The incidence of intracerebral hemorrhage (ICeH) was higher in patients with amphetamine dependence (0.40%), and cocaine-related disorders (0.35%) in comparison to those without SUDs (0.29%) (p < 0.0001). The incidence of subarachnoid hemorrhage (SAH) was higher in patients admitted with alcohol-related disorders (0.27%), amphetamine dependence (0.40%), and cocaine-related disorders (0.35%) when compared to those with no SUDs (0.24%) (p < 0.0001). The prevalence of a history of AIS was higher in patients admitted with cocaine-related disorders (2.22%) in comparison to those with no SUDs (2.14%) (p < 0.0001). There was a higher prevalence of history of ICeH in patients admitted with alcohol-related disorders (0.42%), amphetamine dependence (0.60%), and cocaine-related disorders (0.53%) in comparison to those patients with no SUDs (0.33%) (p < 0.0001). The prevalence of a history of SAH was higher among patients with amphetamine dependence (0.23%), cocaine-related disorders (0.23%), and nicotine dependence (0.18%) in comparison to those without SUDs (0.14%) (p < 0.0001)  and nicotine dependence (0.08%) in comparison to those without SUDs (0.06%) (p < 0.0001). Similarly, the prevalence of angina pectoris was higher in patients with cocaine-related disorders (0.54%), and nicotine dependence (0.25%) in comparison to those with no SUDs (0.19%) (p < 0.0001). The prevalence of a history of myocardial infarction (MI) was higher in patients with cocaine-related disorders (3.60%) and nicotine dependence (5.31%) in comparison to those with no SUDs (3.33%) (p < 0.0001). The incidence (1.71% vs. 1.70%) and prevalence (2.22% vs. 2.14%) of AIS were higher amongst patients with cocaine-related disorders compared to those without SUDs.Multivariable regression showing odds of hospitalizations with new onset of cerebrovascular disorders and cardiovascular amongst SUDs patientsThe odds of having hospitalizations with new onset of AIS were higher in patients with amphetamine dependence , cocaine-related disorders , and nicotine dependence . The odds of having hospitalizations with new onset ICH were higher in patients with amphetamine dependence , cocaine-related disorders , and alcohol-related disorders . The odds of having hospitalizations with new onset SAH were higher in patients with amphetamine dependence , and nicotine dependence . The odds of having hospitalizations with new-onset myocardial infarction were higher in patients with nicotine dependence  and cocaine-related disorders . The odds of having hospitalizations with new-onset angina pectoris were higher in patients with cocaine-related disorders . The odds of having hospitalizations with new-onset atrial fibrillation were higher in patients with alcohol-related disorders   amphetamine and cocaine use were associated with greater odds of ischemic stroke and ICH, (2) cocaine but not amphetamine use was associated with increased odds of angina and acute MI, (3) alcohol use was associated with an increased odds of ICH and atrial arrhythmias, (4) nicotine use was associated with an increased odds of ischemic stroke, acute MI, and sudden cardiac arrest (SCA).Both amphetamine and cocaine are vasoactive agents that exert their effect through catecholaminergic modulation and have been previously associated with both ischemic and hemorrhagic stroke . A retroIn terms of involvement of the cardiovascular system, hospitalization with angina pectoris and acute MI was greater in patients with cocaine use  but not amphetamine use. This is contrasted to the findings by Westover et al. who found a statistically significant association of acute MI with both cocaine and amphetamine use among hospitalizations in Texas . Their sThe increased legalization of marijuana and its rising prevalence has led to increased concern regarding its long-term effects on the cardiovascular system  . Our stuWe found significantly increased odds of hospitalization with ICH in patients with alcohol-related disorders . A prior retrospective study by Casolla et al. demonstrated similar findings with the proposed mechanism being through coagulopathy and platelet dysfunction . In keepTobacco use is a well-established risk factor and has been shown to have a dose-dependent effect on the risk of ischemic stroke, MI, and ruptured cerebral aneurysms -21. Our Strengths and limitationsOur study showed that there is a higher risk of CVDs among patients with SUDs, even though the evidence described in our findings is based on administrative billing codes, producing a decreased overall sensitivity when compared with clinical diagnosis. The data we provided, together with the relevant literature, show the importance of better education about drug use problems and the effects they have on cardiovascular health. While we were able to demonstrate an association of different types of SUDs with different subtypes of CVDs and CeVDs, our study was limited in further delineating the specific doses or frequency of drug uses and their association with CVDs and CeVDs. Moreover, the study was designed to establish association and not causality.In conclusion, our study highlights the significant variability among CVD and CeVD events among patients hospitalized for SUD. Our results may aid in raising the index of suspicion and early management, especially in young adults hospitalized for SUD. Further studies are needed to evaluate other parameters such as the frequency and dose-relationship of different substances and their effect on CVD and CeVD in hospitalized patients.


Aphanomyces invadans, which elicits anti-cancer and anti-microbial activities. To understand the potential GE18 peptide-induced biological effects, an in silico analysis, in vitro (L6 cells) and in vivo toxicity assays (using zebrafish embryo), in vitro anti-cancer assays and anti-microbial assays were performed. The outcomes of the in silico analyses demonstrated that the GE18 peptide has potent anti-cancer and anti-microbial activities. GE18 is non-toxic to in vitro non-cancerous cells and in vivo zebrafish larvae. However, the peptide showed significant anti-cancer properties against MCF-7 cells with an IC50 value of 35.34 µM, at 24 h. Besides the anti-proliferative effect on cancer cells, the peptide exposure does promote the ROS concentration, mitochondrial membrane potential and the subsequent upregulation of anti-cancer genes. On the other hand, GE18 elicits significant anti-microbial activity against P. aeruginosa, wherein GE18 significantly inhibits bacterial biofilm formation. Since the peptide has positively charged amino acid residues, it targets the cell membrane, as is evident in the FESEM analysis. Based on these outcomes, it is possible that the GE18 peptide is a significant anti-cancer and anti-microbial molecule.Small molecules as well as peptide-based therapeutic approaches have attracted global interest due to their lower or no toxicity in nature, and their potential in addressing several health complications including immune diseases, cardiovascular diseases, metabolic disorders, osteoporosis and cancer. This study proposed a peptide, GE18 of subtilisin-like peptidase from the virulence factor of aquatic pathogenic fungus  A peptide-based drug development approach has recently gained global research attention. Peptide or peptide-based drugs are widely used to address several health complications, such as immune diseases, cardiovascular diseases, metabolic disorders, osteoporosis and cancer . About eAspergillus fumigatus, the pathogenic fungal-derived virulence factor has small molecules (SMs), named fumitremorgins, with significant anti-cancer activity  and incubated for 15 min. Post-incubation, 200 μL of pyruvate (21.5 mmol dm−3) was added, and absorbance was measured at 339 nm using an ELISA reader. The percentage of LDH release was calculated [The cytotoxicity of GE18 in MCF-7 cells was established through the LDH assay. Cells  were grown on a six-well plate and treated with the peptide  for 24 h. Cells were examined using an inverted phase-contrast microscope. The results were acquired as pictures. Cells were double-stained using AO and ethidium bromide (EtBr) [The effect of GE18 on the cellular morphology was determined as described . In briee (EtBr) . MCF-7 c5/well) were stained by adding the 40 µM DCFDA solution for 20 min, and then the excess stain was washed with PBS and observed under a fluorescence microscope facilitated with a Cool SNAP-Pro color digital camera . The results were recorded as photomicrographs. The fluorescent intensity of the images was measured by analyzing the images on ImageJ software .The DCFDA assay measured the peptide-mediated influence on ROS generation in cancer cells ,30,31. I5/well) were stained using rhodamine 123 dye (10 µg/mL) for 30 min in dark conditions. Then, the excess stain was washed with PBS, and the results were recorded as images by observing under the fluorescence microscope at 20× magnification. Fluorescent intensity was measured by processing the images in ImageJ software .Whether or not GE18 influences the mitochondrial membrane potential was measured using the rhodamine assay . In brie5/well) were grown as a monolayer on a six-well plate, and the scratch was made with a sterile plastic pipette tip (10 µL). Then, the monolayer cells were washed with PBS to remove the debris, and then the cells were challenged with the peptide. The results were recorded as images which were captured at 0, 12 and 24 h using a bright-field inverted microscope (40× magnification).A wound-healing assay established the ability of GE18 to inhibit the migration of cancer cells . Cells (5/well), which were treated with the peptide for 24 h. tRNA was converted into cDNA according to the ready script cDNA synthesis kit (Sigma). SYBR Premix ExTaq  and Light Cycler 96  were used to perform the gene expression. The primers used are listed in −ΔΔct method.The gene expression analysis has been executed to understand the molecular mechanism(s) by which GE18 elicits anti-cancer effects. A protein concentration in the range of 1 to 5 μg/μL of total protein in the cell lysate or tissue homogenate was required for the gene expression analysis. Using the Trizol method, total RNA was isolated from the MCF-7 cells (4.0 × 10P. aeruginosa. To determine the minimum inhibitory concentration (MIC), a broth micro-dilution assay was performed [P. aeruginosa was centrifuged. The cell pellet, which was resuspended in double-strength nutrient broth, was then challenged with the peptide at five concentrations. After the 3 h incubation, absorbance readings were taken at 625 nm in an ELISA reader.The radial diffusion assay has been performed to conduct a preliminary screen of the peptide for antibacterial activity against multiple bacteria . A totalerformed . BrieflyP. aeruginosa was challenged with the peptide in five concentrations, while untreated bacteria served as the control. The culture was withdrawn at various time points  and plated on a nutrient agar plate, followed by incubation at 37 °C. The number of colonies formed was calculated at log10 CFU/mL to measure the inhibitory efficiency of the peptide.The bactericidal effect of GE18 has been determined by the time kill assay . The midP. aeruginosa cells were treated with GE18 ; untreated cells served as the control, and the gentamicin (1 mg/mL)-treated group served as the positive control. After the challenge, cells were centrifuged at 6000 rpm for 15 min. The supernatant was collected and utilized to quantify the protein concentration by the Bradford assay. The concentration of intercellular components that were released was measured by following Chen et al. and taking the absorbance values at 290 nm using an ELISA reader [A protein leakage assay was performed with slight modifications . For theA reader .P. aeruginosa culture, at a concentration of 1 × 108 cells/mL, was inoculated in a 96-well plate with a 200 μL culture volume. Then, the culture was exposed to the peptide at five different concentrations and incubated for 24 h at 37 °C. The untreated culture was considered as the control, and the gentamicin (1 mg/mL)-exposed group served as the positive control. Post-incubation, bacterial growth was measured. The biofilm formed was fixed with methanol stained with crystal violet, followed by washing the samples dry. Measurements were read at 570 nm.The biofilm inhibition assay measured the peptide’s efficiency to prevent biofilm maturation . BrieflyThe effect of GE18 on bacterial cellular morphology was examined through a field-emission scanning electron microscopy (FESEM) analysis . The logData represent an average of three replicates ± standard deviation (SD). A one-way ANOVA and Tukey’s multiple range test using Graph Pad Prism (Ver.5.0) were applied to determine the statistical significance at the 5% level among all the performed assays.Different approaches have been tested and accepted as anti-cancer therapy. Yet, there is a need for efficient and precision target molecules with minimal adverse effects, for which a peptide-based treatment has been considered as an alternative in cancer therapy. Therapeutic peptides elicit anti-cancer effects by regulating multiple cellular mechanisms that target the cancer cells via tumor angiogenesis suppression, apoptosis, inhibition of distinct internal targets, membrane rupture and immune modulation ,40. AMPsAphanomyces invadans. The protein of interest has been identified from the cDNA sequence of Aphanomyces invadans transcriptome, and the protein has been named on the basis of the conserved domain present in the proteomic sequence. Similarly, the protein named subtilisin-like peptidase from the pathogen Pseudogymnoascus destructans functions as a virulence factor that causes white-nose syndrome in bats [This study has investigated the anti-microbial and anti-cancer activity of the GE18 peptide, which is derived from the subtilisin-like peptidase or serine protease virulence factor of  in bats . The pro in bats ,48.2O2) showed several adverse effects. Further, the effect of GE18, on whether or not it influenced the physiological cellular events such as ROS, apoptosis and lipid peroxidation were assessed. The results, as in Aphanomyces invadans and showed significant anti-cancer properties against the Hep-2 cells [GE18 originated from the virulent factor protein sequence, which should not be toxic to normal cells. Yet, its toxic effects were assessed in both in vitro (L6 cells) and in vivo zebrafish embryo models. The in vitro results showed that the GE18 is non-toxic, which was further established via the developmental toxicity assay on the zebrafish larvae. Zebrafish have several significant advantages over the other in vivo models ,50,51. T-2 cells . The comBrevibacillus laterosporus promotes the increase of LDH in epidermal cancer [Grapsus albacarinous) anti-cancer activity against MCF-7 cells [The in vitro anti-cancer effect of GE18 has been established in breast cancer cells (MCF-7). GE18 is a small molecular weight molecule, >3 kDa, and it has ample charged amino acid residues HIS 1, SER 2, THR 1, GLY 3 and uncharged residues ARG 1, GLU 1, ASP 1, which are the key contributors to GE18′s anti-cancer effect . GE18 shl cancer , wherein-7 cells . GE18 af-7 cells .Callyspongia fstularis (marine sponge) symbiont Bacillus pumilus AMK1 showing a significant increase in the level of ROS [Allium sativum, which induced caspase-3- and caspase-9-mediated apoptosis against leukemic cell lines [GE18-exposed MCF-7 cells upregulated intracellular ROS concentrations. A similar outcome has been reported previously, with a dipeptide from l of ROS . It is al of ROS . Based ol of ROS . Anti-tul of ROS . The genll lines .P. aeruginosa in the radial diffusion and MIC assays. This anti-microbial effect of GE18 is similar to the effect of RM12, a peptide from the Tachykinin protein derivative of Channa striatus [P. aeruginosa. The protein leakage assay (P. aeruginosa, which is in line with the outcome of the RM12 peptide [P. aeruginosa [The anti-bacterial activity of GE18 has been assessed against ten bacteria as a preliminary screening approach. Significant anti-bacterial activity was found against striatus . The bacstriatus . The pepge assay  and relege assay  re-estab peptide . The FESruginosa .A. invadans, is non-toxic. Furthermore, the peptide has significant anti-cancer effects against breast cancer cells and anti-microbial activity against P. aeruginosa. Therefore, we propose that the peptide can be utilized for identifying therapeutical strategies against breast cancer or bacterial infection.Based on the outcomes of the study, we conclude that GE18, derived from the subtilisin-like peptidase or serine protease virulence factor of